tRNA is notoriously difficult to manipulate. Sequencing of tRNA presents various problems because of complex and tightly bound secondary structure and protein association. Despite the difficulties in sequencing tRNAs, the have been extensively sequenced and their structures are well known, see Mathias Sprinzl et al., Compilation of tRNA Sequences and Sequences of tRNA Genes Nucl. Acids Res. (1996) 24 (1): 68-72, hereby incorporated by reference.
Current methods for analysis of tRNAs, and RNA in general, include RNAseq, microarray, and mass spectrometry. These methods are proven tools for detection of novel tRNAs and global tRNA expression patterns (Chan et al. 2011; Dittmar et al. 2006). However, each method has limitations. High throughput RNA sequencing (RNASeq) methods require extensive library preparation, including PCR amplification, to prepare cellular RNA for sequencing. A reverse transcription (RT) step is necessary to copy the original RNA sequence to cDNA, which results in loss of the original RNA strand. Additionally, the RT step is impeded by the occurrence of structure and nucleotide modifications, which are both commonly found in tRNAs. These “RTstops” result in truncated cDNA.
Nanopore sequencing of polynucleotides (including ribopolynuicleotides) works on the principle that when a nanopore is immersed in an ionic solution and a voltage is applied across it, and when a molecule such as a nucleotide passes through (or near) a nanopore, it creates a characteristic perturbation of the current signature passing between two sides of the nanopore. Nanopores may be used to identify individual DNA bases as they pass through the nanopore. Such an approach has been demonstrated and commercialized by Oxford Nanopore Technologies. Using this technology a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification or chemical labelling step.
Nanopore sensors are single molecule based and would allow for examination of several thousand individual tRNA molecules in a single experiment. Biological nanometer scale pores, such as αHL, were proposed as single molecule sensors for nucleic acids almost twenty years ago (Kasianowicz et al. 1996). In concept, the nucleotide sequence of an individual molecule could be read by observing changes in ionic current as the linearized strand is electrophoresed through the nanopore aperture. Recent developments in sensing DNA have coupled an enzyme to regulate DNA movement in single nucleotide steps through a nanopore, which produced ionic current traces that provide a single base readout of DNA sequence (Cherf et al. 2012; Manrao et al. 2012). While no such result has been demonstrated for RNA, work examining immobilized RNA in an engineered αHL pore indicates that an appropriately sensitive nanopore can discriminate between the four canonical RNA nucleotides and specific modified ribonucleotides (Ayub and Bayley 2012).
Experiments by the present inventors and others have shown that DNA cytosine modifications can be detected with high confidence from individual nanopore reads of chemically synthesized DNA (Schreiber et al. 2013; Laszlo et al. 2013). By extension, these results suggest that nanopore sensors could detect sub-molecular features of tRNA, including nucleotide modifications, if tRNAs can be mechanically unfolded and electrically motivated to pass through the pore.
With this in mind the present inventors sought to develop a mechanism to specifically capture tRNA molecules, promote their mechanical unfolding, and initiate threading of the linearized strand through the nanopore lumen.